Recording of odor evoked responses in a genetically defined glomerulus.

A) Schematic diagram illustrating the experimental preparation. A puff of 200 µM methionine was applied during 100 ms to the left olfactory epithelium of Dre.mxn1:GFP Xenopus tropicalis tadpoles. Changes in the Local Field Potential (LFP) were measured ipsilaterally with an electrode targeted to the lateral glomerulus formed by axon terminals of olfactory sensory neurons (OSNs) expressing GFP. The inset shows a confocal projection illustrating labelled OSNs (s: soma, a:axon) and the bilateral formation of glomeruli (g). The Dre.mxn1 promoter also drives GFP expression in the endothelial cells of some blood vessels (e). B) Representative glomerular odor evoked response (black) obtained by averaging individual responses (gray) of the LFP following stimulation (arrow). The asterisk shows a positivity that appeared in 37% of the recordings and preceded the characteristic negativity associated to glomerular activation. C) Negative deflections of LFP (individual responses, gray; average trace, black) were observed when the recording electrode was placed in the GFP labelled glomerulus but disappeared in the mitral cell layer (MC, red traces). D) Experiment showing LFP recordings performed in four different locations of the glomerular layer spaced by 50 μm. The characteristic odor evoked response was observed in only one of the positions tested. The representative glomerular odor evoked response (black) was obtained by averaging individual responses (gray) following stimulation with methionine (arrow). E) Hyperstack projections of the left olfactory bulb of three different Dre.mxn1:GFP Xenopus tropicalis tadpoles. The lateral glomerular cluster (L) is always obvious, and some medial projections (M) are evident in two of the illustrated examples. Color scale indicates dorsoventral disposition.

Odor evoked responses are mediated by glutamatergic neurotransmission.

A) The representative glomerular odor evoked response (black) was obtained by averaging individual responses (gray) of the Local Field Potential (LFP) following stimulation (arrow). In this example, the pipette solution contained 100 µM CNQX and upon local injection of 1 μL, there was a reduction in the amplitude of LFP negativities. B) Mean LFP changes obtained under control conditions (n=21) were reduced after the application of 100 µM CNQX (n=8), 100 µM AP5 (n=5), or both 100 µM CNQX and 100 µM AP5 (n=8). B) Box plot illustrating how the initially recorded peak negativities were affected by the application of 100 µM CNQX, or, 100 µM AP5 together with 100 µM CNQX. Boxes represent the median (horizontal line), 25th to 75th quartiles, and ranges (whiskers) of the indicated experimental groups. Statistical differences were evaluated using paired t-test. C) Rubi-glutamate was injected in the GFP labelled glomerulus and locally uncaged with a 500 ms pulse of blue light (circle). The example shows the change in LFP induced by two flashes (squares) delivered at an interval of 9 seconds. E) Application of picrotoxin, a GABAA antagonist, did not modify odor evoked changes. Recordings show average responses (n=9). Statistical differences were evaluated using paired t-test. F) Odor evoked LFP changes were exclusively triggered by ipsilateral stimulation. Individual responses are indicated in gray, with the representative average response in black. Contralateral stimuli (yellow) did not modify the LFP, as shown in the average representative response (brown).

Potentiation of odor evoked responses by transection of the contralateral olfactory nerve.

A) The number of olfactory sensory neurons (OSNs) and the amplitude of odor evoked negative deflections of the Local Field Potential (LFP) were related to olfactory nerve width according to linear and exponential functions, respectively. Individual data points are represented by circles (n=48). Each bin indicates the mean ± standard error of n=6 tadpoles. The dotted line indicates the steady-state LFP amplitude reached during development. B) Representative odor evoked response (red; individual responses, gray) obtained 24 h after contralateral nerve transection. A solution of 200 μM methionine was used as stimulus (arrow). C) Odor evoked LFP changes exhibit amplitudes above the expected values (dotted line as in A) after contralateral olfactory nerve transection at the indicated time points. The dots represent the mean ± s.e.m obtained 2 to 7 hours (n=10), 1 to 2 days (n=14), and 10 to 11 days (n=11) post-injury. There was a 75% increase in animals recorded 1 to 2 days after transection of the contralateral olfactory nerve (red arrow) compared to control tadpoles (dotted line). D) Dots (mean ± s.e.m.) connected by a line illustrate odor evoked glomerular responses at the indicated times after injury. The superimposed violin plot displays individual data. Most LFPpeak values are above the level expected for the developmental period studied (dotted line as in A).

The potentiation of odor evoked responses is not mediated by injury derived cues.

A) Odor evoked Local Field Potential (LFP) changes were recorded by an electrode targeted to the GFP-positive glomerulus of Dre.mxn1:GFP tadpoles one to two days after bilateral transection of optic nerves. B) Peak LFP negativities recorded in tadpoles with sectioned optic nerves did not exhibit the characteristic potentiation observed after transection of the contralateral olfactory nerve, as they remained within the range of values observed during normal development (solid line, as in Fig. 3A). Bins indicate mean±s.e.m., circles show individual values. C) Imaging of reactive oxygen species (ROS) two hours after transectioning one olfactory nerve (arrow). The ratio between the fluorescence emitted by HyPer-YFP when excited at 488 nm and 405 nm is indicated in pseudocolor. Notice that ROS were increased at the injury site but remained at basal levels in both olfactory bulbs as indicated by the box plot. Each circle shows values collected in a single tadpole. D) Block of ROS production by incubating tadpoles with 200 μM apocynin (n=10) or 2 μM diphenyleneiodonium (DPI, n=5) did not modify the amplitude of odor evoked LFP responses recorded 24 h after contralateral olfactory nerve transection (n=10). Boxes represent the median (horizontal line), 25th to 75th quartiles, and ranges (whiskers) of the indicated experimental groups.

The presynaptic component of glomerular activation is affected by damage to contralateral olfactory sensory neurons.

A) Application of a puff of 200 μM methionine to the olfactory epithelium activates a set of glomeruli in the ipsilateral olfactory bulb (arrows) of tubb2b:GCaMP6s tadpoles. Images show the relative changes in GCaMP6s fluorescence (ΔF/F) obtained after two sequential stimulations carried out in a single tadpole. B) Time course of the responses detected in the glomeruli indicated in A). C) Example showing the simultaneous recording of Local Field Potential (LFP) and changes in GCaMP6s fluorescence in the region targeted by the electrode. Colored traces and gray traces show the change in GCaMP6s fluorescence (ΔF/F) and LFP respectively observed after three sequential applications of 200 μM methionine. Black traces show the average ΔF/F and LFP responses. D) Kinetics of the change in LFP observed in tadpoles with the contralateral olfactory nerve transected between 2 h and 48h prior to recording. E) Intracellular calcium increases detected in glomeruli of control tadpoles with intact olfactory pathways (35 glomeruli, 10 tadpoles, black), and, in tadpoles subjected to the transection of the contralateral olfactory nerve (10 glomeruli, 3 tadpoles, red). Each trace indicates the response of a glomerulus to a single stimulus. Solid lines and error bars indicate mean ± s.e.m. F) Calcium transients detected in tadpoles with an olfactory nerve transected showed a larger amplitude and a rising phase with a shorter time constant (τ). Boxes in D) and F) represent the median (horizontal line), 25th to 75th quartiles, and ranges (whiskers) of the indicated experimental groups. Statistical differences in D) and F) were evaluated using paired and unpaired t-tests, respectively. Circles in D) indicate values obtained for a single tadpole and in F) refer to a single glomerulus.

Contralateral input modulates presynaptic inhibition mediated by dopamine D2 receptors and is involved in the potentiation of glomerular responses.

A) Recordings obtained in a control tadpole showing how the amplitude of Local Field Potential (LFP) responses (gray traces) obtained during a baseline period of 8 minutes increase in a time-dependent manner after local application of 300 nM raclopride, a D2 receptor antagonist. B) Box plot showing the effect of 300 nM raclopride (blue) on the amplitude of LFP responses recorded in tadpoles with full capacity to process odors (control, n=8, black) and tadpoles subjected to the transection of the contralateral olfactory nerve (n=11, red). Boxes represent the median (horizontal line), 25th to 75th quartiles, and ranges (whiskers) of the indicated experimental groups. The effect of raclopride weas evaluated using the paired t-test and the comparison between control and transected groups was performed using the unpaired t-test. C) Relative change in LFP responses induced by 300 nM raclopride in control tadpoles and in tadpoles subjected to the transection of the contralateral olfactory nerve. Dots represent mean ± s.e.m. The solid black line illustrates the fit to a Hill equation, defining a T50 at 20 minutes. D) Simultaneous recording of LFP and changes in GCaMP6s fluorescence in a tubb2b:GCaMP6s tadpole. Gray traces and light blue traces show individual responses to sequential stimulations before and after application of 300 nM raclopride, respectively. Average responses are shown in black and dark blue. E) Application of CGP-36742, a GABAB receptor antagonist, did not modify LFP responses. The box plot compares the amplitude of LFP changes recorded before (gray) and 20 min after local application of 300 μM CGP-36742 (green). Statistical differences were evaluated using paired t-test. E) Time course of relative LFP changes induced by 300 μM CGP-36742. Dots represent mean ± s.e.m (n=10).

Changes in odor evoked responses caused by selective photoablation of olfactory sensory neurons innervating the homologous contralateral glomerulus.

A) Odor evoked Local Field Potential (LFP) changes were recorded one day after the selective elimination of olfactory sensory neurons (OSNs) located in the right nasal cavity. After the identification of GFP-positive OSNs in the epithelium labeled with the nuclear marker Hoechst 33342, regions containing ≥2 fluorescent neurons were identified and photobleached. Cell targeting was confirmed by the suppression of the nuclear label. Only cells found within the photobleached areas exhibited fragmented nuclei (arrows) 24 hours after photobleaching. B) Examples showing odor evoked responses recorded in a tadpole incubated with Hoechst 33342 (black, average), and in a different tadpole 24 hours after the photobleaching of selected regions in the contralateral olfactory epithelium (violet, average). C) Plot of variance against the amplitude of LFPpeaks in three experimental groups of tadpoles: control (n=17), photoablated (n=10) and subjected to unilateral olfactory nerve transection (n=14). Circles show individual values and dots indicate mean±s.e.m. The line shows a fit through values of control and unilaterally transected tadpoles (r=0.65). D) Model proposed for the bilateral modulation of glomerular output in the olfactory bulb of Xenopus tadpoles. Juxtaglomerular neurons display tonic dopamine release that inhibits glomerular output by activating presynaptic D2 receptors present in OSNs (dotted box). The constant presence of dopamine within glomeruli is favored by the activity of the contralateral olfactory bulb. When the contribution of the contralateral pathway is suppressed, dopamine release diminishes, and glomerular responses become potentiated. The contralateral modulation of the tonic activity of dopaminergic juxtaglomerular neurons corrects for input differences and equalizes the synaptic output of olfactory glomeruli to achieve a bilaterally balanced transfer of information. Axons sent by mitral cells could mediate the pathway described but, the involvement of neurons found in higher brain regions and participating in the processing of olfactory information, such as those of the lateral pallium, could also be considered.